Elastic fibers in the tunica propria of the seminiferous tubules. Light and electron microscopic investigations.

Histological examination of numerous biopsies from mature testes, repeatedly showed irregularities of the elastin staining in the tunica propria of the seminiferous tubules. Even when abundant elastic fibers were visible by light microscopy, no elastic fibers were demonstrable in the electron microscope in ultrathin sections of testicular tubules embedded in Epon and contrasted with phosphotungstic acid (PTA). In 43 biopsies from 22 men aged 17-39 years (19 investigations of sterility and three patients with hypogonadotropic hypogonadism) we therefore checked for the occurrence of elastic fibers in the wall of the seminiferous tubules. Matrix loci of elastic fibers could indeed by demonstrated by electron microscopy using PTA and potassium permanganate (KMnO4), but only after embedding in araldite. Under these conditions, light and electron microscopic findings agreed with each other. The appearances of moderate and severe testicular tubular atrophy differed slightly from one another with regard to the amount of elastin. In the "Sertoli cells only syndrome", elastic fibers were demonstrable only outside the hyalinized inner layer. In the Klinefelter syndrome, only "uncertain" elastin loci were present, but greatly increased microfibrils were to be seen using the electron microscope. No elastic elements and only very sparse microfibrils were present in the tunica propria of the tubules of young men with hypogonadotropic hypogonadism.     

Role of elastin anisotropy in structural strain energy functions of arterial tissue

The vascular wall exhibits nonlinear anisotropic mechanical properties. The identification of a strain energy function (SEF) is the preferred method to describe its complex nonlinear elastic properties. Earlier constituent-based SEF models, where elastin is modeled as an isotropic material, failed in describing accurately the tissue response to inflation–extension loading. We hypothesized that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We performed inflation–extension tests on common carotid of rabbits before and after enzymatic degradation of elastin and applied constituent-based SEFs, with both an isotropic and an anisotropic elastin part, on the experimental data. We used transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBFSEM) to provide direct structural evidence of the assumed anisotropy. In intact arteries, the SEF including anisotropic elastin with one family of fibers in the circumferential direction fitted better the inflation–extension data than the isotropic SEF. This was supported by TEM and SBFSEM imaging, which showed interlamellar elastin fibers in the circumferential direction. In elastin-degraded arteries, both SEFs succeeded equally well in predicting anisotropic wall behavior. In elastase-treated arteries fitted with the anisotropic SEF for elastin, collagen engaged later than in intact arteries. We conclude that constituent-based models with an anisotropic elastin part characterize more accurately the mechanical properties of the arterial wall when compared to models with simply an isotropic elastin. Microstructural imaging based on electron microscopy techniques provided evidence for elastin anisotropy. Finally, the model suggests a later and less abrupt collagen engagement after elastase treatment.     

Fluorescence microscopic distinction between elastin and collagen

Summary Distinction between elastin and collagen in arteriosclerotic lesions is difficult because immature and incompletely cross-linked collagen bind so-called elastica stains; furthermore, abnormal collagen can lack cross-striation and thus resemble elastin in electron microscopy. However, collagen and elastin differ significantly in their content of basic amino acids and hence in their affinity for heteropolyacids. This chemical difference was utilized for the development of a fluorescence microscopic method for distinction between collagen and elastin.Paraffin sections of human autopsy material were treated with a 1% aqueous solution of phosphomolybdic acid (PMA) for five minutes, rinsed in distilled water, dehydrated and mounted. Other series were treated with the PMA-molybdenum blue reaction and with various special stains.Only elastic membranes of aorta, the elastica interna and externa of sizable arteries, and true elastic fibers remained strongly fluorescent; the autofluorescence of collagen, reticulum fibers, basement membranes, pseudo-elastic fibers, and elastic membranes in small arteries was quenched. In other series PMA abolished the fluorescence of basic fluorochromes.Correlation of fluorescence and direct light microscopic observations with chemical and electron microscopic data showed that the PMA-fluorescence method permits distinction between elastin and various types of collagen.     

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials

Background

Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.

Methods

Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.

Results

MS² data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.

Conclusions

The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.

General significance

The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.     

Ultrastructure of lung elastin and collagen in mouse models of spontaneous emphysema.

The tight-skin (Tsk) and beige (bg) mutants of the C57B1/6J strain of mouse spontaneously develop air-space enlargement reminiscent of human emphysema. To determine if this enlargement is accompanied by matrix destruction, as in the human disease, we examined the elastin and collagen matrices of the lungs of both mutants. The ultrastructure of these matrix components was separately visualized by scanning electron microscopy following controlled alkali digestion, which preserves collagen, and formic acid digestion, which enables visualization of elastin. Significant elastin destruction suggestive of an elastolytic process was observed in the lungs of Tsk mice. Thickening of elastin lamellae was observed in the lungs of bg mice, suggesting that congenital matrix remodeling may underlie air-space enlargement in this strain.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study

Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view.     

Structure and mechanical properties of the longitudinal ligaments and ligamentum flavum of the spine

Stress-strain curves were recorded from anterior and posterior longitudinal ligaments and ligamenta flava dissected from pig lumbar spines. Ligaments were examined during extension by light microscopy, to observe crimp structure, and by X-ray diffraction, to determine collagen fibril orientations. Scanning electron microscopy (SEM) was used to examine ligaments fixed at high and low strains. Initial stages of ligament extension involvd alignment of collagen fibrils. Collagen fibrils in unstrained ligamentum flavum were much more disoriented than in the longitudinal ligaments. Thus, fibril alignment, and consequent stiffening, occurred at much higher strains than for longitudinal ligaments, allowing ligamentum flavum to exploit the extensibility of its elastin.     

Organization of rat mesenteric artery after removal of cells or extracellular matrix components

Summary Rat mesenteric arteries, perfusion fixed in relaxed or contracted conditions, were digested with acid and elastase, bleach (sodium hypochlorite), or alkali to selectively remove collagen, elastin, or cells. Scanning electron microscopy was used to study the three-dimensional organization of the remaining cells or extracellular components. Smooth muscle cells of the tunica media were elongated and circumferentially oriented. Superior mesenteric artery cells had an irregular surface with numerous projections and some ends were forked. Small mesenteric artery cells were spindle shaped with longitudinal surface ridges, and showed extensive corrugations upon contraction. Elastin was present both as laminae and as an interconnected fibrous meshwork. Collagen was arranged in an irregular network of individual fibrils and small bundles of fibrils that formed nests around the cells in both arteries. This irregular arrangement persisted, with no apparent reordering or loss of order, upon contraction. The lack of an ordered arrangement or specialized organization at the cell ends suggests mechanical coupling of the cells to elastin or collagen throughout the length of the cell, allowing for force transmission in a number of directions. The tunica media is thus a composite material consisting of cells, elastin, and collagen. The isotropic network of fibers is well suited for transmitting the shearing forces placed on it by contraction of smooth muscle cells and by pressure-induced loading.     

Evidence for the intramolecular pleating model of fibrillin microfibril organisation from single particle image analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.     

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.     

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S- GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S- GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.     

Synthesis and structural characterization of poly(LGGVG), an elastin-like polypeptide

Poly(LGGVG) a potential elastin-like biomaterial has been synthesized and studied both in solution (by circular dicroism and nuclear magnetic resonance) and in the aggregated state (by transmission electron microscopy). For sake of comparison, also the conformation of the protected (Boc-LGGVG-OEt) and free (H(2)(+)-LGGVG-OH) 'monomers' has been investigated. While in the latter ones the presence has been evidenced of more or less stable type II beta-turns, the polymer showed a conformational ensemble, possibly comprising type II beta-turns, type I beta-turns and open (unordered) structures. At supramolecular level, twisted-rope aggregates were observed by transmission electron microscopy for the polymer. Thus, the title compound has shown to possess, at both molecular and supramolecular level, physico-chemical properties very similar to those of elastin, so to give some confidence that it could really constitute the precursor of an artificial substitute of elastin itself.     

Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures

Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis. Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin. The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration. Primary hamster lung cell cultures were utilized. Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone. Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine. These cross-links are found only in elastin. Addition of [14C] lysine to cultures results in uptake of the radiolabel into the cross-links. Cross-links were isolated and identified using chromatography and electrophoresis. At all doses of amiodarone, elastin synthesis was seen to increase above control levels. Light and electron microscopy confirmed the presence of an extracellular matrix. The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.     

An engineered minidomain containing an elastin turn exhibits a reversible temperature-induced IgG binding.

A two-helix version of the triple alpha-helical staphylococcal Protein A, previously shown to retain the Fc binding properties of protein A, has been engineered to contain an elastin sequence, GVPGVG, within the inter-helix turn. The original type I beta-turn was replaced with a beta-turn from the muscle protein elastin, which has an inverse temperature-induced folding transition. These "elastin mutants" had lost their helical structure, as measured by circular dichroism (CD), and exhibited a lower stability than the wild-type domains (T(m) reduced by about 48 degrees C) in 30% trifluoroethanol. For the wild-type domains, the amount of alpha-helix and the binding affinity for Fc decreased as the temperature was increased. In contrast, although the starting affinity was lower for the disulfide elastin-turn mutant, it exhibited a 21-fold improvement in affinity over the same temperature range. The melting curve for the elastin-turn minidomain showed cooperative behavior, as measured by the increase in CD-amplitude at 222 nm. The observed CD behavior is consistent with the formation of a type I beta-turn, exhibiting similar DeltaH and DeltaS values to those seen previously for short elastin peptides [Reiersen, H., Clarke, A. R., and Rees, A. R. (1998) J. Mol. Biol. 283, 255-264], and accounting for the increase in on-rate. This demonstrates that, when inserted into a stable globular protein, short elastin sequences have the ability to modify local structure and activity, by operating as temperature modulated switches.     

Expression of elastolytic cathepsins in human skin and their involvement in age-dependent elastin degradation

BackgroundSkin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides.MethodsCathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry.ResultsElastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies.ConclusionCathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.     

Cellular and body scale morphology of Heterocapsa huensis sp. nov. (Peridiniales, Dinophyceae) found in Hue, Vietnam

Cellular and body scale structure of a new armored dinoflagellate Heterocapsa huensis , collected from Hue, Vietnam were investigated. Morphology of motile cell was observed by light, fluorescent and scanning electron microscopy, and body scale structure was examined by whole mounts of transmission electron microscopy. Cells of H. huensis were ellipsoid with a spherical nucleus located in the posterior and multiple pyrenoids located above the nucleus; this arrangement was similar to that of Heterocapsa pygmaea . Transmission electron microscopy revealed ultrastructure of the body scales consisted of a rounded triangular basal plate and three-dimensional ornaments. Structure of the basal plate resembles that of Heterocapsa illdefina ; however, the number of the peripheral spine is different from that of H. illdefina and this structure has never been reported from Heterocapsa species. A new Heterocapsa species, H. huensis Iwataki et Matsuoka sp. nov., is described based on positions of organelles and body scale ultrastructure.     

On the Fine Structure of Valvognathia pogonostoma gen. et sp.n. (Gnathostomulida, Onychognathiidae) with Special Reference to the Jaw Apparatus

Valvognathia pogonostoma gen. et sp.n. is described as belonging to the family Onychognathiidae Sterrer, 1972. An integrated picture of the jaw apparatus is presented, based on interference phase contrast (Nomarski) and electron microscopy. The teeth are shown to be continuous with lamellae, apophyses, fibulae, and even jugum. These structures are all extracellular secretions covering the apical surfaces of the jaw apparatus epithelium. They constitute a single, interconnected, "cuticular" structure. Spiral-ciliary-organs and rhabditoid organelles are described in the epidermis.     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.     

13C CPMAS NMR studies of the elastin-like polypeptide (LGGVG)n

The elucidation of structure-function relationships in insoluble elastin is often approached using elastin-like polypeptides. In this manner, the characterization of the different regions in this extensive biopolymer may be facilitated in a "piece-wise" manner. Our solid-state NMR experiments indicate that (LGGVG)n has structural similarities to elastin and some elastin peptides, providing support for the utility of the mimetic peptides. Furthermore, previous NMR and CD studies indicated that the structure of the elastin-like polypeptide (LGGVG)n in solution is best described as a "conformational ensemble" with a mixture of type I and II beta-turns, in addition to unfolded regions. Our data indicate that the peptide does not adopt a single conformation in the solid state, lending further support to models for elastin that involve significant conformational heterogeneity.